The voltage-sensing domains (VSDs) of voltage-gated ion channels (VGICs) play a crucial role in regulating ion transport across the cell membrane. For decades, clinical trials have been unsuccessful in developing a non-opioid analgesic targeting NaV1.7 despite it being considered a promising drug target for treating chronic pain. Further study of the channel and its gating properties in the presence of ligands may provide new paths for drug development.
Gating modifier toxins (GMTs) are a class of peptides often derived from animal venoms, that potently modulate the function of VGICs. Early studies showed several orders of magnitude differences in functional modulation and binding. The difference in binding affinity and functional activity was assumed to be due to lipid partitioning of the toxins, which was not accounted for in the binding studies performed using ion channels or segments of ion channels solubilised in detergent micelles.
Here, we used cyclised nanodisc to investigate the trimolecular complex, including the toxins, lipids, and segments of ion channels. Our study focused on the NaV1.7-VSD and the prototypical model of this class of channels, the KvAP-VSD from an archaebacterium. Using isothermal titration calorimetry (ITC), we titrated VSTx1, a GMT and potent inhibitor of KvAP, into KvAP-VSD nanodiscs to investigate the GMT binding mechanism. Additionally, we successfully produced the isolated Nav1.7-VSD-DII and its chimera, which can be locked in the resting state by another GMT (HwTx-IV) and assembled them into nanodiscs. The binding affinity of m3-HwTx-IV for the chimera nanodisc was observed in nanomolar range, consistent with previous studies on high-affinity GMT-channel binding in vitro. Our data suggest that lipid partitioning contributes weakly to channel binding and that, instead, the state and dynamics of the toxin binding site on the channel are important determinants that account for differences observed in lipid and detergent environments. Our findings highlight that the high-affinity binding between ligands and channels can be achieved without strong dependence on lipid partitioning. Also, compared to VSD reconstitution in detergent micelles, cyclised lipid nanodiscs help stabilise NaV1.7/Ab-VSD-DII chimera in its correct resting state, which has significant implications for biophysical ligand discovery efforts.